|Title||Optimization of canine interleukin-12 production using a baculovirus insect cell expression system Biotechnology|
|Author(s)||Pinheiro, Cristiane Garboggini Melo De; Pedrosa, Mayara De Oliveira; Teixeira, Naiara Carvalho; Ano Bom, Ana Paula Dinis; Oers, Monique M. Van; Sá Oliveira, Geraldo Gileno de|
|Source||BMC Research Notes 9 (2016). - ISSN 1756-0500|
Laboratory of Virology
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||Baculovirus - Dog - Interleukin-12 - Protein expression optimization|
Background: Interleukin-12 is an important cytokine in mediating cellular immune responses. Results: Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized IL-12 construct contained the GP64 signal peptide and was synthesized with optimized codons for expression in Trichoplusia ni cells. Dot-blot and Western blot analysis showed the highest production levels of recombinant IL-12 protein by the use of the modified baculovirus vector containing the optimized insert, at a multiplicity of infection of five and at 48 h after infection. The recombinant cytokine was successfully purified and showed a good degree of purity, integrity, folding, and yield, with very little endotoxin contamination. Recombinant canine IL-12 induced IFN-γ in canine lymphocytes, indicating that it was biologically active. Conclusion: Therefore, this study describes an efficient method to produce adequate amounts of biologically active canine IL-12, useful for immunomodulation studies in dogs.